Molecular Determinant Underlying Selective Coupling of Primary G-Protein by Class A GPCRs

Abstract

G-protein-coupled receptors (GPCRs) transmit downstream signals predominantly via G-protein pathways. However, the conformational basis of selective coupling of primary G-protein remains elusive. Histamine receptors H₂R and H₃R couple with G_s- or G_i-proteins respectively. Here, three cryo-EM structures of H₂R-G_s and H₃R-G_i complexes are presented at a global resolution of 2.6-2.7 Å. These structures reveal the unique binding pose for endogenous histamine in H₃R, wherein the amino group interacts with E206^5.46 of H₃R instead of the conserved D114^3.32 of other aminergic receptors. Furthermore, comparative analysis of the H₂R-G_s and H₃R-G_i complexes reveals that the structural geometry of TM5/TM6 determines the primary G-protein selectivity in histamine receptors. Machine learning (ML)-based structuromic profiling and functional analysis of class A GPCR-G-protein complexes illustrate that TM5 length, TM5 tilt, and TM6 outward movement are key determinants of the G_s and G_i/o selectivity among the whole Class A family. Collectively, the findings uncover the common structural geometry within class A GPCRs that determines the primary G_s- and G_i/o-coupling selectivity.

Keywords: G protein selectivity; GPCR; H2R; H3R; cryo‐EM structure; machine learning; signaling complex.

Figure 1

Cryo‐EM structures of H₂R‐G_s and H₃R‐G_i complexes. A) (Up) Proportion of G_s‐ or G_i/o‐coupled receptors in the non‐olfactory GPCRs. (Down) Number of receptors primarily coupling with G_s‐ or G_i/o‐proteins. B) The cryo‐EM density map (left) and atomic model (right) of amthamine‐bound H₂R‐G_s complex. The amthamine is depicted as stick within a transparent EM density map. C) The cryo‐EM density map (left) and atomic model (right) of H₃R‐G_i complex. The ligands histamine and immepip are depicted as sticks within a transparent EM density map.

Figure 2

Ligand recognition of histamine receptors. A) Detailed interaction of amthamine with H₂R. Dashed lines represent hydrogen bonds. B) Amthamine induced cAMP accumulation in HEK293 cells expressing H₂R mutants of the residues in orthosteric pocket (n = 3, ordinary one‐way ANOVA, ****P < 0.0001, ND, not determinable, which refers to cannot be established over the tested concentration range, ns refers to no significance between the WT and mutant). C) Structural comparisons of ligand recognition between histamine‐H₁R and amthamine‐H₂R structures. D) Sequence alignment of the orthosteric pocket of histamine receptors. Residues that interact with ligands are highlighted with colored circles. E) Dose‐dependent curves for amthamine induced cAMP accumulation in HEK293 cells expressing the H₂R mutants (V99^3.33Y, D186^5.42T, T190^5.46N) that are not conserved in H₁R (n  = 3). F‐G) Detailed interactions of histamine F) and immepip G) with H₃R. H) Agonists induced G_i dissociation in HEK293 cells expressing H₃R mutants of the residues in orthosteric pocket by NanoBiT assays (n = 3, ordinary one‐way ANOVA, *P = 0.0456, ****P < 0.0001, NR refers to no response to the ligand, ND, not determinable, which refers to cannot be established over the tested concentration range, ns refers to no significance between the WT and mutant).

Figure 3

Endogenous ligand binding poses of aminergic receptors. A–E) Binding poses of H₃R (A), H₁R (B), D1R (C), 5‐HT_1A (D), and β₁AR (E) with its endogenous ligands. F) Superimposition of H₃R‐hisatime and H₁R‐histamine structures. G,H) Effects of histamine‐induced G‐protein dissociation in HEK293 cells expressing histamine receptor mutants at 5.46 position. Dose‐response curves for H₃R E206^5.46 mutants (G) and H₁R N196^5.46E mutant (H) were measured using the NanoBiT assay (n  = 3).

Figure 4

Structural comparison of histamine receptor–G‐protein complexes. A) Structural superposition of the active models of H₁R, H₂R, and H₃R. B) Comparison of TM5 and TM6 in H₁R‐G_q, H₂R‐G_s, and H₃R‐G_i complexes. C,D) Surface (left) and cartoon (right) representation of binding area of H₂R (C) and H₃R (D) with their respective coupled Gα subunit. Residues within 4Å of H₂R or H₃R in Gα_s or Gα_i are highlighted in yellow or orange, respectively.

Figure 5

TM5 and TM6 are responsible for G_s and G_i selectivity. A) Representative of the TM5/TM6 structural geometry of G_s‐coupled receptors. B) Representation of the TM5/TM6 structural geometry of G_i‐coupled receptors. C) Violin plots depicting four distinct features of TM5/TM6 in G_s‐ and G_i/o‐coupled receptors. The white dot denotes the median. The interquartile range is shown by the broad black bar in the middle. Except for points considered to be “outliers” using an interquartile range‐based technique, the thin line reflects the remainder of the distribution. n = 54, ^ns P > 0.05, *P < 0.05, **P < 0.01 by Mann‐Whitney U test. D) Schematic representation of the “TM5/TM6 swap” experiments. E–F) Dose‐dependent curves for histamine induced G‐protein dissociation and cAMP accumulation in HEK293 cells expressing chimera receptors (n  = 3). E) Replacement of I^5.56‐F^6.44 of H₂R with F^5.56‐F^6.44 of H₃R did not confer the ability to dissociate G_i‐protein (left), but resulted in loss of the ability to activate the G_s signal pathway (right). F) Replacement of P^5.50‐P^6.50 of H₃R with P^5.50‐P^6.50 of H₂R resulted in gain of the ability to activate the G_s signal pathway (left), but loss of the ability to dissociate G_i‐protein (right).

Figure 6

Machine learning‐based classification of GPCRs into G_s and G_i signaling pathway. A) Workflow for GPCR classification utilizing machine learning and feature pre‐processing. The length and tilt of TM5, as well as the length and outward movement of TM6 are extracted from active GPCR structures obtained from GPCRdb homology models and True structures. The resulting data of these four geometries are standardized, and then subjected to PCA. Subsequently, a Random Forest classifier is employed to classify GPCRs based on the PCA results. B,C) PCA biplots were generated for the four geometries of GPCRdb homology models (B) and True GPCR structures (C) individually. The contributions of each variable to the principal components are depicted as vectors on the plot, where the vertical component of a vector on a given PC illustrates the respective contribution of that variable to the PC. The angle between two vectors reflects the correlation between the corresponding features, while the length of a vector represents the significance of the corresponding feature. D) Comparison of violin plots depicting four geometries of TM5/TM6 in GPCRdb structures and True GPCR structures. Here the median and interquartile range are depicted as dashes. E) Confusion matrix for the True GPCR structures given by the best classifier. F) Decision boundary visualization via PCA given by the best classifier and scatter plot of GPCRdb homology models (left) and True GPCR structures (right) based on GPCRdb homology models.

Figure 7

Contributions to G‐protein signaling of extended TM5/TM6. A) (Up) Surface and cartoon representations of Gα_s are shown, with the surface of Gα_s depicted as transparent. (Down)The binding surface of TM5 with Gα_s of D1R is shown, with TM5 beyond I^5.69 noted as extended helix. Residues that interact with Gα are highlighted in green. B) (Up)Surface and cartoon representations of Gα_i are shown, with the surface of Gα_i depicted as transparent. (Down) The binding surface of TM6 with Gα_i/o of 5‐HT_1A. TM6 beyond R^6.29 was considered as extended helix. C) The additional contact areas of G_s‐ or G_i/o‐coupled receptors between the extended TM5/TM6 with Gα_s/Gα_i/o, respectively. D) Schematic representation of the TM5‐ (up) or TM6‐ (down) breaking mutants. E) Dose‐dependent curves for histamine induced cAMP accumulation or G‐protein dissociation in HEK293 cells expressing the truncated receptors (n  = 3). (Up) The TM5 of H₂R was respectively truncated by four amino acids and eight amino acids. The cAMP assay validated that the length of TM5 was closely related to the activation of the G_s signal pathway. (Down) The TM6 of H₃R was respectively truncated by eight amino acids and nine amino acids. The NanoBit assay validated that the length of TM6 did not correlate with the dissociation of the G_i‐protein. F) Schematic representation of the geometry determinants for G‐protein selectivity of GPCRs.